Four G protein coupled receptor, OGR1, GPR4, G2A and TDAG8, belong to a novel subfamily of receptors. We have recently identified that these receptors are the first and only receptors for sphingosylphosphorylcholine (SPC) and lysophosphatidyicholine (LPC), which are important bioactive lipid signaling molecules. Each of these receptors, share some similar, but distinct, binding and activating features. We propose that the specific amino acid residues reside in 7TMs are responsible for ligand binding and the different intracellular domains in OGR1 family receptors that are required for their shared as well as specific signaling activities. This hypothesis will be tested in three specific aims. Specific Aim 1: Identify the amino acids required for SPC and LPC binding in OGR1 subfamily genes and construct three-dimensional (3D) models of OGR1 and G2A. Mutational and functional analyses (binding and activity assays) will be performed to identify the structural determinants for ligand binding. Molecular models of OGR1 and G2A will be constructed based on structure-function data generated. Specific Aim 2: Determine the amino acid residues required for antagonist binding in TDAG8 and the mechanisms of PMA-induced OGR1 desensitization. The structural determinants for antagonist binding in TDAG8 will be identified using mutational studies. The mechanism for PMA-induced desensitization will be determined through mutational and functional studies. Specific Aim 3: Determine the structural requirements for activating the calcium and MAP kinase signaling pathways in OGR1 subfamily genes. Chimeric receptors will be made between OGR1 and other receptors to determine the structural element(s) that is required for differential coupling to Gi and Gq proteins, leading to the increase in intracellular calcium; the structural elements responsible for the lack of coupling to the calcium pathway and which are essential for MAP kinase activation. The involvement of SPC and LPC in many diseases, such as arteriosclerosis, inflammatory diseases and cancers makes these receptors highly attractive targets for the treatment of these diseases. The structure-function relationship data to be obtained through proposed studies will lay the foundation for future development of new agonist(s) and/or antagonist(s) for OGR1 family receptors, which are crucial for establishing the physiological and pathological roles and functions of these receptors, and eventually the development of drugs.